DELIGNIFIED CELLULOSIC MATERIALS TO IMPROVE INDUSTRIAL PROCESSES OF ALCOHOLIC FERMENTATION

Abstract: A method for the removal of lignine from cellulosic materials is proposed. Such materials can be used as catalysts in fermentation for alcohol production, by using glucose or molasses solutions as raw materials. With the methods used and the results obtained, it was shown that lignine free materials increase the rate of alcoholic fermentation as well as the final alcohol content, compared to the fermentation without such materials. The usage of lignine free materials is also proposed for the immobilisation of fermentation yeast cells. Biocatalysts produced in such a way were shown to be suitable for the production of wine and beer at low temperatures (0-5 DEG C) yielding satisfactory fermentation times, but also for fermentation at normal temperatures with low fermentation times and for a large number of fermentation batches. Finally, the biocatalyst that resulted from cells immobilisation in lignine-free cellulosic materials, proved to be suitable for wine and beer production in a continuous operation reactor. This was shown by the fact, that the reactors productivity was not reduced following its operation for 65 days.

Description: O 95/ DESCRIPTION DELIGNIFIED CELLULOSIC MATERIALS TO IMPROVE INDUSTRIAL PROCESSES OF ALCOHOLIC FERMENTATION This invention aims to the use of delignified cellulo materials as catalyst of alcoholic fermentation and supports f immobilization of the yeast cells. Likewise, the biocatalys prepared by immobilisation of yeast on delignitied cellulos 5 materials are suitable for low temperature alcoholic termentati as well as for continuous processes. After the oil crisis of 70 decade, the alcohol fermentation becomes more interested since bioethanol was thoug 10. that can be used as a renewable fuel for automobiles. Howeve the energy demand and production cost of bioethanol will have be reduced significantly in order this fuel successfully be us and in contrast to benzin. There fore for reducing cost t research was focused (i) to increase the productivity 15 bioreactors and (ii) to the reduction of the energy demand of t production plant. The first, researchers have been attempted obtain by the cell immobilization on solids supports, so that possible the use of the continuous bioreactors. Last yea attempts were made to employ substances increase the ethan 0 production rate. The second, " the researchers have been attempt to achieve by the increase of V. v/v alcohol concentration in t product of fermentation. However, in order to be used t immobilization of cells in industry as prerequisites are t abundance of the support in nature and its low cost. 5 Increase of the fermentation rate give cellulosic materia such as sawdust and much more increase was obtained by t delignifed cellulosic materials which was prepared after remov of lignin. This removal was achieved after the treatment of wat 0 sawdust with NaOH IV. solution at the b.P. of the water. electron microscope studies was indicated that significant por in the mass of cellulose was obtained which increased i treatment with hot water. Likewise, delignified product augmen SUBSTITUTESHEET the final ethanαl concen rations at higher values than those are obtained by non delignified cellulosic material and much more higher of that achieved by the fermentation of liquids carried out without delignified or non delignified cellulosic materials. Therefore, in the case which one use delignified cellulosic material as promoting material at higher initial sugar concentrations the ethanol production yield is higher than the case of without of it. It is supported to lower residual sugar analyzed and higher final ethanol concentration obtained and the treatment with hot sodium hydroxide solution increases the possibility cells going through and so accommodate their immobilization. That in relation with low prise of the support and its abundance in nature make this biocatalyst attractive in the ethanol production as well as in wine making and beer production. Also, the particles form of the support give the possibility for fermentations using feed batch bioreactors and separation of biocatalyst employing centrifuge separators or separation after removal of the supernatant liquid. The last will be achieved only in the case which it is possible. The form of particles of the support permits the use of a fluidized bed bioreactor. These bioreactors can be used in the potable alcohol production, grade - fuel alcohol production, wine making and beer production. The immobilization of yeast cells on dedignified cellulosic materials has been studied by electron microscope as well as by the possibility of this biocatalyst prepared to ferment from batch to batch. It is well known that fermentations of must at relatively low temperatures are carried out with low rates and they are stopped before the end of the total sugar consumption. There fore, high residual sugar concentrations leads to alterations of wine. In the case which one can prepares wine at low temperatures with completed fermentations and without any alteration, the wine being prepared have improved aroma and taste. Wine making with fermentations at lower of 10 t are very difficult. Likewise, wine making with fermentations at 0-4 °C in a level of alcohol content 12-15'/. v/v have not carried out and relative results are SUBSTITUTE SHEET not found in the literature. Thus, the development of a process to increase fermentation rate at low temperatures makes these fermentations more interested and the dangerous they not to be completed takes off. Furthermore, the discovering of a fermentation process at 0-4 °C for wine making and beer production is an important contribution to their production. Biocatalyst prepared by immobilization of the psychrophile and alcohol resistant strain VISANTO - 1 on delignified cellulosic material leads to the production of beer and wine at 0-4 "C with a satisfactory fermentation rate and in a fermentation near of equal to that of natural fermentation of must at room temperature. This biocatalyst give an important increase of the fermentation rate. The last leads to a reduction of cost and more safe fermentation at low temperatures. I. Method for preparation of delliαnified cellulosic material. In a beaker of 5 1 contains 3 1 of NaOH solution was added 300 gr of sawdust powder. It was heated for 3 hours at the boiling point so that the volume remains constant by the addition of water. After that, it was filtrated and the cellulosic material prepared washed many times with hot water. The wet solid pressed on the Buchner funnel to remove the water be possible. Also, 680 g with 65"/. humidity were prepared. This product was stored in a refrigerator and used (i) for the experiments of the promotion of alcoholic fermentation and (ii) as support for immobilization of yeast cells used in the alcoholic fermentation in the temperature range 0-30 °C. II. Alcohol fermentation of glucose in the presence of delignified cellulosic material. Two conical flasks of 11 contain each 300 ml synthetic media containing glucose 40"/. [ initial "Be density 19.4 ], 0.5"/. MgS0 and 0.4 yeast extract in it were also diluted 0.1 KH 2 PQ as well as 0.17. (NH,^S0 4 . In this liquid was spread baker's yeast so SUBSTITUTE SHEET that to obtain on cell concentration 20 g/1. In the first flas. was not added any amount of delignified cellulosic material, while in the second was added so that the concentration of this material in a dry weight basis be equal to 80 g/1. However, since delignified cellulosic material contains 65"/. humidity, it causes a reduction of glucose and cell concentration. In order concentrations be constant, the appropriate amounts of glucose ard baker's yeast were added. The flasks were incubated at 25 °C for fermentation. The results are presented below. Parameter Blank With delignified (without deligni cellulosic fied cellulosic material material) Final "Be density 6.5 Final alcohol 12.8 1 .3 concentration "/.v/v E t h a n o l 17 25 productivity (g/l/d) III Molasses alcoholic fermentation in the presence of delignified cellulosic material. Two pairs of conical flasks of 1 1 contained each 400 ml of rerified molasses and the ° Be density of it was to one pair of Flasks 16 and to other 18.2. To all samples were spread baker's yeast so that the final cell concentrations be constant, appropriates amounts of molasses and baker's yeast were added. Two of them were contained delignified cellulosic material 80 SUBSTITUTE SHEET g/1. In the following, flasks were incubated at 27°C and allowe for fermentation. Results are given below. Para- Without With Without With meter delignified delignified delignified delignified eellulosic eellulosic cellulosic cellulosic material material material material Initial "Be 16 16 18.2 18.2 Final Be 6.4 10.2 6.9 Final alcohol concentration 7. (v/v) 9.6 10.4 9.4 11.3 Fermentation time (hr) 70 36 95 57 Immobilization of yeast cells on delignified cellulosic material . In a synthetic media containing glucose of 800 ml with pH=4.8 and 12"_ glucose was spread an amount of biomass obtained by culture of the strain AXA2-1 of Saccharomyces cerevisial species. Furthermore, 170 g delignified cellulosic material were added and incubated for fermentation. Before the completion of fermentation the supernatant was decanted and the rest was filtrated using a Buchner Funnel. Then the biocatalyst was washed two time with synthetic media containing glucose and after filtration pressed on the funnel to remove the water. After that delignified cellulosic material supported biocatalyst prepared directly for wine making with repeated batch fermentation was used. SUBSTITUTE SHEET V. Repeated batch fermentation far wine making at room and low temperatures. In a glass cylinder of 1 1 contained grape must or synthetic media containing glucose was added delignified cellulosic material supported biocatalyst prepared as above described by immobilization αf cells on delignified cellulosic material. The cylinder was incubated for different experiments at 30, 16, 10, 0 °C. Before the completion of fermentation samples were filtrated using a Buchner funnel washed three time with must of .synthetic media containing glucose. Then, the biocatalyst pressed on the funnel and used for the next fermentation batch. Thus, was obtained 55 repeated batch fermentations. Fermentations performed at 10, 16, 30 "C gave wines containing 11.2 - 15.37. v/v ethanol at 17 - 33 hr fermentation time. At the fermentations of 0 "C, wines obtained with 12.1 - 15.67. v/v ethanol at 665 - 2640 hr. VI. Repeated batch fermentations for beer production at low temperatures. In a glass cylinder of 1 1 containing must extract an amount of biocatalyst prepared by cell immobilization on delignified cellulosic material was added. Glass cylinder was incubated successively at 10, 5 and 0 °C. Just before the end of fermentation the liquid was filtrated an a buchner funnel, washed three time with must extract and every time the biocatalyst was pressed on the funnel and the wet solid was used for the next fermentation batch. Thus, all the above operation was repeated for 10 repeated batch fermentations. For the temperature of 10 °C were prepared beer produces with 5 - 5.57. v/v ethanol concentrations at 100 - 300 hr. fermentation time. VII. Continuous wine making In a cylindrical bioreactor (height 33 cm and 12 cm diameter), placed in vertical position in a constant temperature of 25 °C water bath, were added 500 g wet delignified cellulosic SUBSTITUTE SHEET material. Into bioreactor was also added synthetic media containing 127. glucose and remained for fermentation for 4 hr. Then, was pumped for 2 days by a high accuracy peristaltic pump with the synthetic media containing glucose having the same composition. After that, it was pumped with must of 12 "Be density and flow rate in the range of 500 - 1100 ml/day. Total acidity of grape must used was changed successively from 4 to 6 and then to 8 of trartraric acid /l. The Bioreactor was pumped for 65 days with must without any substantial decrease in the productivity. Products obtained every day had a 10.7 -11.27. v/v alcohol concentrations, residual sugar 25 - 40 g/1 and volatile acidity 0.15 - 0.35 g CH,00H/ 1 SUBSTITUTE SHEET

Claims: CLA IMS We c l aim : 1. The possession of the right concern the alcoholic fermentation method of sugar solutions using free or immobilized cells, which is characterized by the presence of delignified cellulosic material as catalyst of as support for immobilization of cells. The method includes in relation with the use of delignified cellulosic material, repeated or not batch fermentations for the potable and grade - fuel alcohol production, wine making and brewing in the range of temperatures of which fermentation can carried out, or production of these products in continuous bioreactors. 2. In relation with claim 1 the alcoholic fermentation of glucose solutions in the presence of delignified cellulosic materials with or without of one or more nutrient in order to produce potable of grade - fuel alcohol. 3. In relation with claim 1 molasses alcoholic fermentation in t e presence of delignified cellulosic material to increase fermentation rate and . final ethanol concentration for the production of potable and grade - fuel alcohol. 4. In relation with claim 1 the immobilization on delignified cellulosic material of yeast cells or other microorganism makes alcoholic fermentation. 5. In the relation with claims 1 and 4 the use of biocatalyst (prepared by immobilization on delignified cellulosic material of yeast cells or other microorganism makes alcoholic fermentation) for wine making at room and low temperature with repeated or not batch fermentations. 6. In the relation with claims 1, 4 and 5 the use of biocatalyst (prepared by immobilization on delignified cellulosic SUBSTITUTE SHEET material of yeast cells) for brewing at low temperatures and temperatures at which usually brewed, using repeated or not batch fermentations. 7. In the relation with claims 1, 4, 5, 6 the use of biocatalyst (prepared by immobilization on delignified cellulosic material of yeast cells of other microorganism makes alcoholic fermentation) for continuous wine making and brewing or for continuous wine making from which the wine will be used as raw material for distillates and potable alcohol production. SUBSTITUTE SHEET

Inventors: BARDI EUSTRATIA, ICONOMOU LAZAROS, KOUTINAS ATHANASIOS, BARDI, EUSTRATIA, ICONOMOU, LAZAROS, KOUTINAS, ATHANASIOS
Applicants: BARDI EUSTRATIA, ICONOMOU LAZAROS, KOUTINAS ATHANASIOS, BARDI, EUSTRATIA, ICONOMOU, LAZAROS, KOUTINAS, ATHANASIOS
Application year: 1994
Main classification: C12N11
IPC codes: C12C11, C12G1, C12M1, C12N11, C12P7